Understanding the agronomic performance and genetic diversity of sweet and bitter lupin genotypes is crucial for developing improved varieties with enhanced yield, nutritional quality, and disease resistance. This study evaluated the agronomic performance and molecular diversity of five sweet (Accessions 1, 2, 3, 4, and 5) and five bitter (‘Giza 1’, ‘Giza 2’, ‘Giza 3’, Accession A, and Accession B) lupin (Lupinus albus L.) genotypes across two growing seasons (2021-2022 and 2022-2023). Significant differences were observed among genotypes for phenological, growth, and yield-related traits. Accession-A consistently demonstrated superior performance across most measured traits, including plant height, number of branches and pods per plant, seed number per plant, 100-seed weight, and seed yield. ‘Giza-1’ and Accession-B also exhibited promising agronomic characteristics. Clustering analysis based on agronomic data categorized genotypes into three groups, with Accession-A forming a distinct group due to its high performance. Principal component analysis revealed strong positive correlations between yield and several agronomic traits, while days to flowering showed a negative association. Molecular analysis using start codon targeted (SCoT) markers revealed polymorphism among the genotypes, with Accession-A and Accession-B exhibiting the highest similarity. Phylogenetic analysis based on SCoT markers clustered the genotypes into four groups, reflecting the genetic diversity among them. Biochemical analysis revealed that sweet lupin (as Accession 5) demonstrated superior total protein content, phenolic, and flavonoid levels. Sweet lupin also had enhanced antioxidant activity, as in Accessions 5 and 3, achieving up to 93% scavenging of DPPH free radicals. Both sweet and bitter lupin displayed significant antibacterial activity, but sweet lupin, as Accessions 5 and 3, showed the highest inhibition zones against Staphylococcus aureus and Escherichia coli. These findings provide valuable information for lupin breeding programs, particularly for selecting superior genotypes like Accession-A and understanding the genetic relationships among different lupin genotypes.